Hookworm infection remains one of the greatest public health threats worldwide, with an estimated 800 million people infected. Heavy hookworm infection is the leading cause of anemia in the tropics, resulting in debilitating and sometimes fatal iron-deficiency anemia caused by blood loss to feeding adult worms in the intestine. Children, pregnant women, and the elderly are particularly susceptible to morbidity from hookworm infection. Control strategies are restricted to periodic de-worming of infected individuals, which is limited by rapid re-infection rates and the development of drug resistant worm populations. Vaccine efforts suffer for the lack of effective target antigens. Development of new drug targets and improved vaccine antigens will require a better understanding of hookworm biology, particularly the infective process. However, the obligate requirement for a host and the inability to grow the complete life cycle in vitro precluded development of powerful genetic tools for hookworm research until recently. We propose to adapt and build upon recent advances using other parasitic nematodes to develop a transfection system for hookworms. We will take two parallel but independent approaches to develop transient transfection methods for hookworms that will provide a foundation for the future development of heritable transformation technology. In Aim 1, particle bombardment will be used to introduce reporter genes into embryos and larval stages, and transformants will be assayed for survival, growth and reporter gene expression. We will also use a recently described antibiotic selection technique to select transformants from non-transformants. In Aim 2, we will use chemical and lipid-based technology to introduce reporter genes into molting larvae, a technique that was recently successful in the parasitic nematode Brugia malayi. In both aims we will use a piggybac retrotransposon-based integrating vector to introduce the reporter transgenes into hookworm chromosomes. Development of in vitro transfection would represent a significant advance for hookworm research, and will allow determination of hookworm gene expression and function in a homologous genetic context for the first time. Furthermore, transgenesis would enable us to exploit the data from the imminent release of several sequenced hookworm genomes and transcriptomes for the development of novel hookworm control strategies.